Lyme disease is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group. It is a zoonotic disease affecting humans, dogs, horses and other mammalian species. The bacteria are transmitted to the mammalian hosts by infected ticks (Ixodes spp.). Lyme disease is the most common vector-borne disease in the United States, Europe and Asia. In Europe and Asia the disease is commonly caused by B. garinii and B. afzelii, while in the United States B. burgdorferi sensu stricto strains are present. In current methods for diagnosis of Lyme disease, serum antibodies to whole B. burgdorferi lysates or to individual antigens of the spirochete are commonly analyzed to identify dogs and horses that were exposed to the pathogen and are at risk of developing disease. In dogs and horses, the detection of serum antibodies to B. burgdorferi can be performed by ELISA followed by Western blotting (WB), which is an inadequate procedure that is nevertheless still considered the gold standard for human Lyme disease diagnostics. While certain tests are available (such as snap tests for detecting the invariable domain IR6 of the variable surface antigen VlsE of B. burgdorferi for dogs and horses), they lack a desirable level of sensitivity and cannot distinguish between various stages of the disease. Thus, there is an ongoing and unmet need for improved methods for diagnosing Lyme disease in mammals, including but not necessarily limited to horses and dogs. The present invention meets these and other needs.